Jean-Christophe Pagès

ICE inhibitor YVADcmk is a potent therapeutic agent against in vivo liver apoptosis

In liver, apoptosis is a physiological process involved in the clearance of injured cells and in homeostatic control [1]. However, in patients with viral fulminant hepatitis or with nonacute liver diseases [2], dramatic liver failure or secondary cirrhosis results from the death of hepatocytes, which express the cell-surface receptor Fas, by apoptosis. To date, treatment of fulminant hepatitis relies mainly on orthotopic liver transplantation, which is limited by immunological complications and graft availability. Unravelling the molecular mechanisms that underlie acute liver failure could allow the design of an appropriate therapy. Ligand-bound Fas and tumour necrosis factor alpha (TNF-alpha) induce hepatic apoptosis in mice [3-6]. In various cell types, Fas- or TNF-alpha-induced apoptosis is blocked by viral proteins (such as p35 and CrmA) as well as by a decoy peptide (YVADcmk) [7-11], suggesting that these mechanisms of apoptosis involve ICE (interleukin-1 beta converting enzyme)-like proteases. Here, we report that, in vivo, pre-treatment of mice with YVADcmk protects them from the lethal effect of anti-Fas antibody and from liver failure induced by injection of TNF-alpha. Remarkably, YVADcmk administration is also highly effective in rescuing mice that have been pretreated with anti-Fas antibody from rapid death, despite extensive hepatic apoptosis. This dramatic curative effect could be of clinical benefit for the treatment of viral and inflammatory liver diseases.

KCa2.3 channel-dependent hyperpolarization increases melanoma cell motility.

Cell migration and invasion are required for tumour cells to spread from the primary tumour bed so as to form secondary tumours at distant sites. We report evidence of an unusual expression of KCa2.3 (SK3) protein in melanoma cell lines but not in normal melanocytes. Knockdown of the KCa2.3 channel led to plasma membrane depolarization, decreased 2D and 3D cell motility. Conversely, enforced production of KCa2.3 protein in KCa2.3 non-expressing cells led to the plasma membrane becoming hyperpolarized, and enhanced cell motility. In contrast, KCa3.1 channels had no effect on cell motility despite an active role in regulating membrane potential. Our data also suggest that membrane hyperpolarization increases melanoma cell motility and that this occurs through the KCa2.3 channel. Our findings reveal a previously unknown function of the KCa2.3 channel, and suggest that the KCa2.3 channel might be the only member of the Ca(2+)-activated K(+) channel family involved in melanoma cell motility pathways.

Autocrine Fibroblast Growth Factor 18 mediates dexamethasone-induced osteogenic differentiation of murine mesenchymal stem cells

The potential of mesenchymal stem cells (MSC) to differentiate into functional bone forming cells provides an important tool for bone regeneration. The identification of factors capable of promoting osteoblast differentiation in MSCs is therefore critical to enhance the osteogenic potential of MSCs. Using microarray analysis combined with biochemical and molecular approach, we found that FGF18, a member of the FGF family, is upregulated during osteoblast differentiation induced by dexamethasone in murine MSCs. We showed that overexpression of FGF18 by lentiviral (LV) infection, or treatment of MSCs with recombinant human (rh)FGF18 increased the expression of the osteoblast specific transcription factor Runx2, and enhanced osteoblast phenotypic marker gene expression and in vitro osteogenesis. Molecular silencing using lentiviral shRNA demonstrated that downregulation of FGFR1 or FGFR2 abrogated osteoblast gene expression induced by either LV‐FGF18 or rhFGF18, indicating that FGF18 enhances osteoblast differentiation in MSCs via activation of FGFR1 or FGFR2 signaling. Biochemical and pharmacological analyses showed that the induction of phenotypic osteoblast markers by LV‐FGF18 is mediated by activation of ERK1/2‐MAPKs and PI3K signaling in MSCs. These results reveal that FGF18 is an essential autocrine positive regulator of the osteogenic differentiation program in murine MSCs and indicate that osteogenic differentiation induced by FGF18 in MSCs is triggered by FGFR1/FGFR2‐mediated ERK1/2‐MAPKs and PI3K signaling. J. Cell. Physiol. 224: 509–515, 2010.

Event report: SynBio Workshop (Paris 2012) – Risk assessment challenges of Synthetic Biology

In Europe and beyond, several advisory bodies have been monitoring the developments in the field of Synthetic Biology. Reports have been sent to national governments for information on the developments and possible regulatory and risk assessment questions raised by this field. To put the issues in a broader perspective, four national biosafety advisory bodies (the French High Council for Biotechnology, the German Central Committee on Biological Safety, the Netherlands Commission on Genetic Modification and the Belgian Scientific Institute of Public Health (Biosafety and Biotechnology Unit)) decided to join forces and organize an international scientific workshop to review some of the latest scientific insights and look into possible challenges in the risk assessment of Synthetic Biology. The SynBio Workshop (Paris 2012) – Risk assessment challenges of Synthetic Biology took place on the 12th of December 2012 and gathered scientists from biosafety advisory bodies from fifteen European countries, from the European Food Safety Authority as well as representatives of the European Commission, together with research scientists selected for their excellence in the field. The workshop was divided into two sessions: the first session gave an overview of four major fields in Synthetic Biology. The second session was set up for discussion with a scientific panel and the audience to identify and address relevant questions for risk assessment raised by recent and future developments of Synthetic Biology. An overview of the workshop and the discussion points put forward during the day are discussed in this document.

538. Transient Non-Viral RNA Delivery Mediated by a Lentiviral Particle

Safe and efficient gene therapies including gene-targeting technologies are very challenging but very promising approaches nowadays. The scientific and clinical communities have been working for a long time together to encounter substantial clinical advances they have made possible thanks to numerous improvements in cell culture and gene transfer methods. Opportunities to improve gene transfer into primary or stem cells involve a better design of the vectors used. Such improvements must lead to an increase of the transduction efficiency including the percentage of positive cells, as well as a better level and duration of expression, cell phenotype preservation and the number of genes delivered.. Lentiviral vectors have seen their use largely increased in clinical protocols over the past few years but safety concerns have been highlighted. First, the permanent genetic modification remains a focus of significant regulatory oversight and even integrase- or reverse transcriptase-deficient lentiviral vectors leads to residual integration events. Moreover, all the gene-editing technologies entail a “hit-and-run” mechanism that requires only a transient expression of the nuclease complex. In parallel, mRNA delivery is a versatile, flexible, and safe mean for protein therapies but chemical or electroporation-based transfection protocols are known to induce cell toxicity and phenotype modifications of the target cells. Here, we describe a new chimeric lentiviral platform that allows mRNA delivery into the target cells without any genomic signature. The respective properties of the MS2 bacteriophage and the lentiviral vectors have been combined to build a non-integrative packaging system in which the wild type HIV packaging sequence is replaced by the MS2 stem-loop repeats and the MS2 Coat sequence is inserted into the NucleoCapsid sequence. The resulting lentiviral particle is able to deliver a non-viral RNA into the cytoplasm of target cells, directly available for protein translation. Transduction of immortalized cells but also of T cells and HSC with these RNA lentiviral particles (RLP) shows an efficient, fast and transient expression of both reporters and functional proteins such as genome editing enzymes. Particles structure and functionality, cell transduction and characterization of such engineered cells have been compared with those obtained with an integrative lentiviral vector. Particularly by recruiting the RNA independently of dimerization with more than four molecules per particles, RLPs allow the cotransfer of different species of RNA into target cells. This new delivery system is a great candidate handle the safe and clinically suitable delivery of the editing machinery, which can transiently act without inducing cellular toxicity or immunogenicity.

Retroviral Gene Transfer for LDL Receptor Deficiency into Primary Hepatocytes

Although the pharmacological approach has proven its efficacy for most diseases there are still situations where praticians remain helpless. Among these, genetic diseases are the more difficult to deal with as correction of the defect requires the precise targeting of a protein. As far as the liver is concerned, genetic lesions may affect two kinds of products, excreted proteins and cellular proteins. Whereas the latter need to be produced in the affected cell the former could be delivered by subcutaneous injection or produced by a nonphysiological organ easier to target (myoblast, fibroblast..). Gene therapy is the most attractive therapy as it restores function in a stable manner by introducing alongsides the dysfunctional gene its wildtype counterpart (1). In recent years, systems have been developed for gene transfer. Of particular interest are recombinant viruses among which retroviruses and adenoviruses are the more widely used.